1. Field of the Invention
The present invention relates generally to the fields of surgery, cancer and histopathology. More particularly, it concerns a method for identification of lymph nodes and the presence or absence of lymph node metastases as an important prognostic factor in early stage cancers of all types. In particular aspects, the present invention relates to carbon particle dye compositions for sentinel lymph node identification in cancers such as melanoma. In other aspects, the present invention relates to carbon particle dye compositions for use in determining the histopathologic status of sentinel lymph nodes.
2. Description of Related Art
Routine lymphadenectomy for patients with clinical stage I melanoma remains controversial. Since the first description of a technique for intraoperative lymphatic mapping and sentinel lymphadenectomy (LM/SL) (Morton et al., 1990; Morton et al., 1992), the histologic status of the sentinel node has become a widely accepted criterion upon which to base a decision for complete lymph node dissection in melanoma (Morton, 1997; Thompson et al., 1995).
A large number of studies using the mature technique of LM/SL to identify sentinel nodes with blue dye and radiocolloid support the concept that the histopathologic status of the sentinel nodes is representative of the histopathologic status of all the lymph nodes present in the same basin (Morton et al., 1993; Reintgen et al., 1994; Thompson et al., 1995; Krag et al., 1995; Pijpers et al., 1995; Albertini et al., 1996; Joseph et al., 1997; Bostick et al., 1999; Leong et al., 1997; Loggie et al., 1997; Lingam et al., 1997; Thompson et al., 1997; van der Veen et al., 1994). Unfortunately, a low but definite recurrence risk exists in the same basin after LM/SL is performed as the solitary procedure for tumor-free sentinel nodes (Essner et al., 1999; Gershenwald et al., 1998; Miliotes et al., 1996).
Recurrence in the operated basin that may be due to three reasons. First, in-transit lymphatic metastasis in evolution at the time of LM/SL may subsequently seed the basin. Unfortunately, surgeons presently have no control over this biologic phenomenon.
Second, LM/SL is fallible because a false-negative rate exists due to a failure of surgery, lymphoscintigraphy or histopathology (Morton and Chan, 1999; Morton and Bostick, 1999). Isosulfan Blue and Patent Blue V dyes are the most commonly used agents to identify the sentinel lymph node(s). Surgeons experienced in sentinel node mapping for melanoma have reported successful identification of the sentinel node using blue dye alone in up to 96% of cases (Morton et al., 1992; Morton et al., 1993; Kelley et al., 1998), and the technique is being applied to breast cancer and other solid neoplasms (Giuliano et al., 1997; Morton et al., 1998; Bilchik et al., 1998).
However, isosulfan blue dye-directed mapping is subject to error. The relatively rapid washout of dye from the sentinel node to successive nodes in the basin can lead to intraoperative misidentification of the sentinel node (Bostick et al., 1997; Bostick et al., 1999). This problem led to the use of radionucleotide tracers to assist in identification of the sentinel node. The tracer, usually a technetium-labeled sulfur or albumin colloid, passes through lymphatics into lymph nodes. The sentinel or xe2x80x9chotxe2x80x9d node is then identified by use of a gamma counter. Unfortunately, there is still no clear definition of a sentinel node when using radionucleotide tracer technology (Morton et al., 1998). In patients with breast cancer, Krag et al. (1993) defined a sentinel node as having greater than 25 counts per 10 seconds. Veronesi et al. (1997) defined a sentinel node as having between 10 and 2000 counts per second. Others have defined the sentinel node as a lymph node having ten times as many counts as an adjacent, nonsentinel node (Albertini et al., 1996). As should be evident, these arbitrary standards can lead to confusion and misjudgement.
All blue nodes and/or all radioactive sentinel nodes may not be removed at LM/SL. Time-dependent drainage of radiotracer into multiple nodes has been reported (Glass et al., 1998). This allows non-sentinel nodes to become radioactive, which may mislead the surgeon to remove non-sentinel nodes but inadvertently leave behind the true sentinel nodes (Glass et al., 1998; Morton and Bostick, 1999). Thus, lymph nodes that are declared to be sentinel nodes by the surgeon may not be the true sentinel nodes; however, precise examination of these supposedly sentinel nodes may find them truly histopathologically negative. Additionally, an inherent difficulty with pathologic evaluation of sentinel nodes is that nodes identified and declared by the surgeon as sentinel nodes cannot be histopathologically confirmed, unless metastases are present.
Third, histopathologic evaluation of sentinel nodes may erroneously label sentinel nodes as tumor-negative when micrometastases are actually present. Neither isosulfan blue or radiocolloid is retained in sentinel nodes after processing, and so these agents cannot be identified by light microscopy. This histopathologic shortcoming of LM/SL could be mitigated if the sentinel nodes were stained with a mapping agent that remains in the tissue after histopathologic processing. Upon re-evaluation of sentinel nodes by sectioning and immunohistochemical staining of additional levels in patients with recurrent nodal melanoma after LM/SL, it is often the case that micrometastases were unappreciated at the initial examination (Miliotes et al., 1996). Therefore, any measure which can decrease the false negative rate or the histopathological error rate would potentially decrease the same basin recurrence after removal of tumor-free sentinel nodes.
Thus, there exists a need for improved methods for sentinel lymph node mapping. These improved methods should not exhibit the rapid washout of dyes or the time-dependent drainage of radiotracers. In addition, improved methods of histopathological confirmation of sentinel nodes and identification of tumor cell micrometastases in the sentinel node following lymphadenectomy is needed to promote better survival among this subset of patients.
The present invention describes materials and methods for the identification of sentinel lymph nodes by the surgeon and the pathologist and the presence or absence of lymph node metastases as an important prognostic factor in early stage cancers of all types. The present invention further defines a region of the sentinel lymph node that is identifiable by the surgeon and pathologist and is most likely to contain metastases.
The invention first provides a method of identifying a disease-associated lymph node in an excised tissue sample, comprising, administrating to a subject at least one fluid composition comprising of from about 0.1% carbon particles to about 6.0% carbon particles, excising at least one tissue sample suspected of comprising at least one lymph node, identifying a lymph node by the accumulation of said carbon particles, and; identifying, diagnosing, staging or predicting the presence of neoplastic tissue in said lymph node. As used herein certain embodiments, xe2x80x9cfluidxe2x80x9d means a liquid composition, such as a solution, a suspension, an emulsion and the like. However, in particular aspects, a suspension of carbon particles is preferred. In certain embodiments, the concentration of the carbon particles may vary. In specific aspects, the carbon particle concentration may be about 0.10%, about 0.15%, about 0.20%, about 0.25%, about 0.30%, about 0.35%, about 0.40%, about 0.45%, about 0.50%, about 0.55%, about 0.60%, about 0.65%, about 0.70%, about 0.75%, about 0.80%, about 0.85%, about 0.90%, about 0.95%, about 1.00%, about 1.10%, about 1.20%, about 1.30%, about 1.40%, about 1.50%, about 1.60%, about 1.70%, about 1.80%, about 1.90%, about 2.00%, about 2.10%, about 2.20%, about 2.30%, about 2.40%, about 2.50%, about 2.60%, about 2.70%, about 2.80%, about 2.90%, about 3.00%, about 3.10%, about 3.20%, about 3.30%, about 3.40%, about 3.50%, about 3.60%, about 3.70%, about 3.80%, about 3.90%, about 4.00%, about 4.10%, about 4.20%, about 4.30%, about 4.40%, about 4.50%, about 4.60%, about 4.70%, about 4.80%, about 4.90%, about 5.00%, about 5.10%, about 5.20%, about 5.30%, about 5.40%, about 5.50%, about 5.60%, about 5.70%, about 5.80%, about 5.90%, to about 6.0%, and any range derivable therein. As used herein, xe2x80x9cany range derivable thereinxe2x80x9d means a range selected from the numbers described in the specification. For example, in non-limiting examples, the carbon particle concentration range may be of from about 0.15% to about 5.0%, about 0.15% to about 4.0%, about 0.15% to about 3.0%, about 0.15% to about 2.0%, about 0.15% to about 1.0%, about 0.2% to about 1.0%, about 0.3% to about 1.0%, about 0.4% to about 1.0%, or about 0.5% to about 1.0%.
In other embodiments, the size of the carbon particle may vary. In specific aspects, the carbon particle size (i.e., average diameter) may be about 0.10, about 0.15, about 0.20, about 0.25, about 0.30, about 0.35, about 0.40, about 0.45, about 0.50, about 0.55, about 0.60, about 0.65, about 0.70, about 0.75, about 0.80, about 0.85, about 0.90, about 0.95, about 1.00, about 1.10, about 1.20, about 1.30, about 1.40, about 1.50, about 1.60, about 1.70, about 1.80, about 1.90, about 2.00, about 2.10, about 2.20, about 2.30, about 2.40, about 2.50, about 2.60, about 2.70, about 2.80, about 2.90, about 3.00, about 3.10, about 3.20, about 3.30, about 3.40, about 3.50, about 3.60, about 3.70, about 3.80, about 3.90, about 4.00, about 4.10, about 4.20, about 4.30, about 4.40, about 4.50, about 4.60, about 4.70, about 4.80, about 4.90, about 5.00, about 5.10, about 5.20, about 5.30, about 5.40, about 5.50, about 5.60, about 5.70, about 5.80, about 5.90, to about 6.0 microns, and any range derivable therein. For example, in specific aspects, the carbon particle size range may be of from about 0.1 and about 6.0, about 0.2 to about 4.0, about 0.2 to about 2.0, about 0.2 to about 1.0, or about 0.3 to about 0.8 microns in diameter. In other aspects, the carbon particle size range is less than about 0.2 microns in diameter.
In certain embodiments, the carbon particles comprise carbon black. In specific aspects, the carbon particles may comprise, but are not limited to, channel black, thermal black or furnace black. In other aspects, the carbon black particles are neutral, acidic or basic.
In some preferred embodiments, the composition further comprises at least one additional compound. In specific aspects, the at least one additional compound is a dye. In some aspects, the dye may comprise an anionic dye. In certain aspects, the dye may comprise, but is not limited to, is an acid dye, a basic dye or a direct dye. In particular facets the direct dye may comprise, but is not limited to, Paper Yellow GG (CI Direct Yellow 131), Direct Scarlet 4BS (CI 29160), Congo Red (CI 22120), Violet BB (CI 27905), Direct Sky Blue 5B (CI 24400), Pentamine, Phthalocyanine Blue (CI 74180), Black G (CI 35255) or Deep Black XA (CI Direct Black 154).
In particular facets, the dye may comprise Tartrazine (CI 19140), Quinoline Yellow (CI 47005), Eosin (CI 45380), Acid Phloxine (CI 45410), Erythrosine (CI 45430), Sunset Yellow FCF (CI 15985), Acid Violet 5B (CI 42640), Patent Blue AF (CI 42080), Brilliant Cyanine 6B (CI 42660), Acid Brilliant Blue FCF (CI 42090), Naphthalene Green VSC (CI 44025) or Acid Blue Black 10B (CI 20470). In other facets, the dye is isosulfan blue, guajazulen blue, patent blue V, pentamine or Direct Sky blue, or other dye which travels through the lymphatic system.
In other embodiments, the total concentration of the non-carbon particle, at least one dye, may vary. In specific aspects, the total concentration of dye(s) in the composition may be about 0.10%, about 0.15%, about 0.20%, about 0.25%, about 0.30%, about 0.35%, about 0.40%, about 0.45%, about 0.50%, about 0.55%, about 0.60%, about 0.65%, about 0.70%, about 0.75%, about 0.80%, about 0.85%, about 0.90%, about 0.95%, about 1.00%, about 1.10%, about 1.20%, about 1.30%, about 1.40%, about 1.50%, about 1.60%, about 1.70%, about 1.80%, about 1.90%, about 2.00%, about 2.10%, about 2.20%, about 2.30%, about 2.40%, about 2.50%, about 2.60%, about 2.70%, about 2.80%, about 2.90%, about 3.00%, about 3.10%, about 3.20%, about 3.30%, about 3.40%, about 3.50%, about 3.60%, about 3.70%, about 3.80%, about 3.90%, about 4.00%, about 4.10%, about 4.20%, about 4.30%, about 4.40%, about 4.50%, about 4.60%, about 4.70%, about 4.80%, about 4.90%, about 5.00%, about 5.10%, about 5.20%, about 5.30%, about 5.40%, about 5.50%, about 5.60%, about 5.70%, about 5.80%, about 5.90%, about 6.0%, about 6.10%, about 6.20%, about 6.30%, about 6.40%, about 6.50%, about 6.60%, about 6.70%, about 6.80%, about 6.90%, about 7.00%, about 7.10%, about 7.20%, about 7.30%, about 7.40%, about 7.50%, about 7.60%, about 7.70%, about 7.80%, about 7.90%, about 8.00%, about 8.10%, about 8.20%, about 8.30%, about 8.40%, about 8.50%, about 8.60%, about 8.70%, about 8.80%, about 8.90%, about 9.00%, about 9.10%, about 9.20%, about 9.30%, about 9.40%, about 9.40%, about 9.50%, about 9.60%, about 9.70%, about 9.80%, about 9.90%, to about 10.00%, and any range derivable therein. In other embodiments, the total concentration of the non-carbon particle, at least one dye, may vary in molar concentration. In specific aspects, the total molar concentration of dye(s) in the composition may be about 0.10 mM, about 0.15 mM, about 0.20 mM, about 0.25 mM, about 0.30 mM, about 0.35 mM, about 0.40 mM, about 0.45 mM, about 0.50 mM, about 0.55 mM, about 0.60 mM, about 0.65 mM, about 0.70 mM, about 0.75 mM, about 0.80 mM, about 0.85 mM, about 0.90 mM, about 0.95 mM, about 1.00 mM, about 1.10 mM, about 1.20 mM, about 1.30 mM, about 1.40 mM, about 1.50 mM, about 1.60 mM, about 1.70 mM, about 1.80 mM, about 1.90 mM, about 2.00 mM, about 2.10 mM, about 2.20 mM, about 2.30 mM, about 2.40 mM, about 2.50 mM, about 2.60 mM, about 2.70 nM, about 2.80 mM, about 2.90 mM, about 3.00 mM, about 3.10 mM, about 3.20 mM, about 3.30 mM, about 3.40 mM, about 3.50 mM, about 3.60 mM, about 3.70 mM, about 3.80 mM, about 3.90 mM, about 4.00 mM, about 4.10 mM, about 4.20 mM, about 4.30 mM, about 4.40 mM, about 4.50 mM, about 4.60 mM, about 4.70 mM, about 4.80 mM, about 4.90 mM, about 5.00 mM, about 5.10 mM, about 5.20 mM, about 5.30 mM, about 5.40 mM, about 5.50 mM, about 5.60 mM, about 5.70 mM, about 5.80 mM, about 5.90 mM, about 6.0 mM, about 6.10 mM, about 6.20 mM, about 6.30 mM, about 6.40 mM, about 6.50 mM, about 6.60 mM, about 6.70 mM, about 6.80 mM, about 6.90 mM, about 7.00 mM, about 7.10 mM, about 7.20 mM, about 7.30 mM, about 7.40 mM, about 7.50 MM, about 7.60 mM, about 7.70 mM, about 7.80 mM, about 7.90 mM, about 8.00 mM, about 8.10 mM, about 8.20 mM, about 8.30 mM, about 8.40 mM, about 8.50 mM, about 8.60 mM, about 8.70 mM, about 8.80 mM, about 8.90 mM, about 9.00 mM, about 9.10 mM, about 9.20 mM, about 9.30 mM, about 9.40 mM, about 9.40 mM, about 9.50 mM, about 9.60 mM, about 9.70 mM, about 9.80 mM, about 9.90 mM, to about 10.00 mM, and any range derivable therein. In a non-limiting example, the total dye concentration of the composition may be of from about 0.1 to about 10 mM. Of course, more than one dye may comprise the total dye concentration of the composition.
In preferred facets, the dye is isosulfan blue. In certain facets, the concentration of isosulfan blue is about 0.10%, about 0.15%, about 0.20%, about 0.25%, about 0.30%, about 0.35%, about 0.40%, about 0.45%, about 0.50%, about 0.55%, about 0.60%, about 0.65%, about 0.70%, about 0.75%, about 0.80%, about 0.85%, about 0.90%, about 0.95%, about 1.00%, about 1.10%, about 1.20%, about 1.30%, about 1.40%, about 1.50%, about 1.60%, about 1.70%, about 1.80%, about 1.90%, about 2.00%, about 2.10%, about 2.20%, about 2.30%, about 2.40%, about 2.50%, about 2.60%, about 2.70%, about 2.80%, about 2.90%, to about 3.00%, and any range derivable therein. In a non-limiting example, isosulfan blue is about 0.1% to about 1.0%. In another example, the concentration of isosulfan blue is about 0.25% to about 1%. In other examples, the concentration of isosulfan blue is about 0.5% to about 0.9%. In other embodiments, the composition comprises carbon dye, radiolabeled sulfur colloid and isosulfan blue dye.
In certain embodiments, the at least one additional compound comprises a diagnostic aid. In preferred aspects, the diagnostic aid is Fluorescein or Fluorescein Sodium.
In other embodiments, the at least one additional compound is a radionucleotide tracer. In specific facets, the radionucleotide tracer is technetium-labeled sulfur or albumin colloid, antimony chloride, or other colloidal radionucleotide that travels through the lymphatic system. In some embodiments, the at least one additional compound is a receptor binding compound, an antibody or a locator.
In certain embodiments, the administering of the composition is to the lymphatic region surrounding a neoplastic tissue. In specific facets the neoplastic tissue is a melanoma, lung carcinoma, neuroblastoma, pheochromocytoma, colon, prostate, renal carcinoma, breast carcinoma, esophageal, gastric, pancreatic, oropharyngeal cancer or another neoplasm that metastasizes by the lymphatic channels. In preferred aspects, the neoplastic tissue is a melanoma or a breast carcinoma. In specific facets, the mode of administration is subcutaneous, intramuscular, intralesional, intradermal, intraperitoneal, parenteral, oral, nasal, buccal, rectal, vaginal or orthotopic. In other facets, the time between administering and detecting the carbon particles is about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 7 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about 75 minutes, about 80 minutes, about 85 minutes, about 90 minutes, about 100 minutes, about 110 minutes, about 120 minutes, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours (about 1 day), about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, about 40 hours, about 41 hours, about 42 hours, about 43 hours, about 44 hours, about 45 hours, about 46 hours, about 47 hours, to about 48 hours (about 2 days), and any range derivable therein. In a non-limiting example, the range of time between administering and detecting the carbon particles is between about 5 minutes and about 60 minutes. In another example, the range of time between administering and detecting the carbon particles is between about 1 minute and about 2 days.
In certain embodiments, the subject is an animal, such as a mammal. In preferred aspects, the subject is a human.
In certain embodiments, the tissue sample is removed by a lymphadenectomy. In other embodiments, a lymph node is further identified by using a hematoxylin-eosin histopathological technique, an immunohistochemical technique, spectroscopy or a cancer staging technique. In certain aspects, the method further comprises a microscopic examination of the lymph node.
In particular embodiments, the identifying a lymph node identifies at least one sentinel lymph node. In some aspects, the identifying a sentinel lymph node further comprises histopathology. In certain facets, the histopathology further comprises assessment of carbon particle accumulation in a subregion of the sentinel lymph node. In other facets, the histopathology further comprises identification, diagnosing, staging, or predicting the presence of neoplastic tissue in the sentinel lymph node. In particular facets, a subject who has evidence of micrometastasis in the sentinel node undergo a subsequent lymphadenectomy.
The invention also provides a method of identifying a disease-associated lymph node in an excised tissue sample, comprising, administrating to a subject at least one fluid composition comprising of from about 0.1% carbon particles to about 6.0% carbon particles and isosulfan blue, excising at least one tissue sample suspected of comprising at least one lymph node, identifying a lymph node by the accumulation of said carbon particles, and; identifying, diagnosing, staging or predicting the presence of neoplastic tissue in said lymph node.
The invention provides a method of identifying a disease-associated lymph node in an excised tissue sample, comprising, administrating to a subject at least one fluid composition comprising of from about 0.1% carbon particles to about 6.0% carbon particles and isosulfan blue, excising at least one tissue sample suspected of comprising at least one lymph node, identifying a lymph node by the accumulation of said carbon particles, and; identifying, diagnosing, staging or predicting the presence of neoplastic tissue in said lymph node.
The present invention further describes a method for identifying a disease-associated sentinel lymph node in a subject comprising the steps of administering a carbon particle suspension in combination with a second compound to the lymphatic region surrounding a neoplastic tissue and locating the sentinel lymph node by detecting the accumulation of carbon particles in conjunction with the second compound. In preferred embodiments, the second compound is a dye or a radionucleotide tracer. The dye can be isosulfan blue, guajazulen blue, patent blue V, pontamine or skyblue, or other dyes which travel through the lymphatic system. The radionucleotide tracer can be a technetium-labeled sulfur or albumin colloid, antimony chloride, or other colloidal radionucleotides that travel through the lymphatic system. In preferred embodiments the subject with the neoplastic tissue is human.
Also described are methods and compositions wherein the detecting the accumulation of carbon particles occurs in the sentinel nodes removed by a lymphadenectomy. This can be combined with histopathology of neoplastic tissue and associated lymph nodes. In preferred embodiments, the histopathology further comprises identification, diagnosing, staging, or predicting the presence of neoplastic tissue in the regional or sentinel lymph node. In preferred embodiments, the histopathology further comprises assessment of carbon particle accumulation in a subregion of the sentinel lymph node.
The present invention also describes a method of identifying a disease-associated lymph node in an excised tissue sample following administration of a carbon particle suspension to a subject comprising identifying the sentinel lymph node by the accumulation of said carbon particles and identifying, diagnosing, staging or predicting the presence of neoplastic tissue in said sentinel lymph node. In preferred embodiments, the histopathology further comprises assessment of carbon particle accumulation in a subregion of the sentinel lymph node.
As used herein the specification, xe2x80x9caxe2x80x9d or xe2x80x9canxe2x80x9d may mean one or more. As used herein in the claim(s), when used in conjunction with the word xe2x80x9ccomprisingxe2x80x9d, the words xe2x80x9caxe2x80x9d or xe2x80x9canxe2x80x9d may mean one or more than one.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.